Fig. 1: mPFC^CRF1+ neurons comprise a distinct glutamatergic population that regulates anxiety and conditioned rewarding effects of ethanol.

A Coronal mouse brain atlas [80] image of medial prefrontal cortex (mPFC), corresponding 4X magnification, brightfield and GFP fluorescence images of boxed region depicting the mPFC and 40X magnification, brightfield and GFP fluorescence images depicting neighboring GFP positive (green asterisk; mPFC^CRF1+) and negative (white asterisk; mPFC^CRF1−) mPFC prelimbic (PrL) neurons. B Representative in situ hybridization images of colocalization of Crhr1 (red), Slc17a7 (green), Gad2 (yellow), and DAPI (blue) in the mPFC PrL layer 2/3 subregion and summary graph of percent nuclei expressing. Scale bar = 10 µM. 4 images from N = 2 male mice. C Representative voltage traces of action potential firing elicited by increasing current injections (−120, 40, 60 and 200 pA, 1 s) in mPFC PrL CRF1 non-expressing (mPFC^CRF1−) and CRF1 expressing (mPFC^CRF1+) pyramidal neurons. Average number of action potentials elicited by increasing current injections in mPFC^CRF1− and mPFC^CRF1+ neurons with significant effects of current injection F(29,1972) = 87.27, p < 0.0001; group F(1,68) = 4.26, p = 0.04; and interaction between current injection and group F(29, 1972) = 1.99, p = 0.001 by two-way ANOVA from n = 31–39 cells and N = 10 male mice. D Representative traces of spontaneous excitatory post-synaptic currents (sEPSCs) recorded with a hold potential of −70 mV. E, F Average sEPSC frequency and amplitude in mPFC^CRF1− (black) and mPFC^CRF1+ (green) neurons. n = 19–21 cells from N = 8 male mice; *p < 0.05 by t-test. G Schematic of viral strategy for caspase-mediated ablation of mPFC CRF1-expressing (mPFC^CRF1+) neurons in a Cre-dependent manner in CRF1:Cre mice (Caspase). H Representative in situ hybridization images depicting Crhr1 (green) and DAPI (blue) expression in the mPFC of control (top) and caspase (bottom) mice. Quantification of Crhr1 positive nuclei in mPFC PrL layer 2/3 of control and caspase mice relative to control group. N = 3 male mice/group; **p < 0.01 by t-test. Scale bar = 10 µM. I Latency to feed in an open arena (open bars) and home cage (filled bars) in novelty-suppressed feeding test in control (black) and mPFC^CRF1+ ablated (red) mice with significant main effects of condition F(1,23) = 70.78, p < 0.0001 and group F(1,23) = 4.66, p = 0.04 by two-way ANOVA, and post hoc significance ^#p < 0.05 represented in panel. J Percent time spent in the ethanol paired context during the pre-conditioning phase (open bars) and following ethanol conditioning (filled bars) in the ethanol place conditioning test in control (black) and mPFC^CRF1+ ablated (red) mice with a significant interaction effect F(1,24) = 11.05, p = 0.002 by two-way ANOVA, and post hoc significance ^#p < 0.05 represented in panel. K Pre-conditioning normalized percent time spent in the ethanol-paired side with significant *p < 0.05 effects by one-sample t-test and unpaired t-test. N = 11–15 male mice.